Plant-derived formulations for treatment of hiv

ABSTRACT

Disclosed herein are methods for treating mammals with HIV/AIDS comprising administering a therapeutically effective amount of a composition comprising an extract of  Rubia cordifolia . The extract is preferably an alcohol extract, an aqueous extract, or mixtures thereof. The alcohol preferably comprises ethanol, isopropyl alcohol, or mixtures thereof.

BACKGROUND

HIV (formally known as HTLV-III and lymphadenopathy-associated virus) isa retrovirus that is the cause of the disease known as AIDS (AcquiredImmunodeficiency Syndrome), a syndrome where the immune system begins tofail, leading to many life-threatening opportunistic infections. HIV hasbeen implicated as the primary cause of AIDS and can be transmitted viaexposure to bodily fluids. In addition to percutaneous injury, contactwith mucous membranes or non-intact skin with blood, fluids containingblood, tissue or other potentially infectious bodily fluids pose aninfectious risk. Infection of human CD-4+T-lymphocytes with an HIV virusleads to depletion of this cell population, resulting in animmunodeficient state, and eventually opportunistic infections,neurological dysfunctions, neoplastic growth, and ultimately death.There are currently 18 drugs licensed and used for the treatment of HIV,and these drugs are divided into one of four classes depending on howthey attack HIV. Drugs in the class of nucleoside/nucleotide reversetranscriptase inhibitors are AZT (zidovudine, Retrovir), ddI(didanosine, Videx), 3TC (lamivudine, Epivir), d4T (stavudine, Zerit),abacavir (Ziagen), and FTC (emtricitabine, Emtriva). Drugs in the classof non-nucleoside reverse transcriptase inhibitors are efavirenz(Sustiva) and nevirapine (Viramune). Drugs in the class of proteaseinhibitors are lopinavir/ritonavir (Kaletra), indinavir (Crixivan),ritonavir (Norvir), nelfinavir (Viracept), saquinavir hard gel capsules(Invirase), atazanavir (Reyataz), amprenavir (Agenerase), fosamprenavir(Telzir), and tipranavir (Aptivus). Only one drug is available in theclass of fusion inhibitor, T20 (enfuvirtide, Fuzeon). The antiretroviraldrugs are usually combined into three-drug cocktails called highlyactive antiretroviral therapy or HAART. However, the above-mentioneddrugs still cannot effectively treat AIDS.

Rubia cordifolia, often known as Common Madder or Indian Madder, is aspecies of flowering plant in the coffee family, Rubiaceae. It has beencultivated for a red pigment derived from roots.

International Publication No. WO 2005/030232 describes compositionprepared by extracting pulverized powder from various plants to treatHIV and AIDS. U.S. Patent Application Publication No. 2009/0011052describes a composition including extract powders from medicinal herbsto treat AIDS.

SUMMARY

Disclosed herein is a method of treating a person with HIV/AIDScomprising administering to the person a therapeutically effectiveamount of a composition comprising an extract of Rubia cordifolia. Inone embodiment the composition consists of an extract of Rubiacordifolia. The composition can further comprise apharmaceutically-acceptable carrier or excipient.

The extract can be an aqueous extract, an alcohol extract, or a mixtureof both. Preferably, the alcohol comprises ethanol, isopropyl alcohol,and mixtures thereof.

The extract can prepared using water, organic solvent, or a mixturethereof. Preferred organic solvents are low molecular weight alcohols,halogenated hydrocarbons, organic ethers, low molecular weight esters,low molecular weight ketones, and mixtures thereof.

BRIEF DESCRIPTION OF THE FIGURES

For a more complete understanding of the disclosure, reference should bemade to the following detailed description and accompanying drawingswherein:

FIG. 1 is graph comparing the p24 antigen concentration at variousconcentrations of an ethanol extract of Rubia cordifolia;

FIG. 2 is a graph comparing the transcript accumulation index for theHIV-1 LTR gene at various concentrations of an ethanol extract of Rubiacordifolia; and

FIG. 3 is graph comparing the suppression of the HIV-1 LTR geneexpression with 10 nM Azidothymidine and various concentration of theextract of Rubia cordifolia.

DETAILED DESCRIPTION

Disclosed herein is a method for treating a person with HIV/AIDScomprising administering to the person a therapeutically effectiveamount of a composition comprising an extract of Rubia cordifolia.

An extract of Rubia cordifolia can be prepared in many ways, includingalcohol extracts or other organic solvent extracts. A preferred extractis an alcohol extract of Rubia cordifolia. An alcohol extract of Rubiacordifolia can be prepared by extracting dry root powder of Rubiacordifolia with 75% v/v ethanol. For example, a 10 mg/ml sample can beprepared by suspending 10 mg dry root powder of Rubia cordifolia in onemL of 75% v/v ethanol, followed by rigorous vortexing. The suspensioncan be allowed to stand at room temperature for at least 2 hours andintermittently mixed with a vortex mixer. The supernatant can becollected as the alcohol extract of Rubia cordifolia. Alcohols used toprepare the extract can include low molecular weight alcohols, such asmethanol or ethanol, or mixtures of low molecular weight alcohols withwater. Organic solvents used to prepare the extract can includehalogenated hydrocarbons, organic ethers, low molecular weight esters,low molecular weight ketones, other organic solvents, and combinationsthereof.

The dry root powder of Rubia cordifolia can include collecting driedplants parts of Rubia cordifolia and refluxing the plant parts usingwater, organic solvent, or a mixture thereof. The refluxed plant partscan be filtered and the liquid collected. The liquid can beconcentrated, and then dried to form a dry powder of Rubia cordifolia.Dried plant parts of Rubia cordifolia can include the leaves, root, orcombinations thereof.

In some embodiments, the composition consists of an extract of Rubiacordifolia. The phrase “consists of” excludes other compounds that haveanti-HIV activity. Thus, the composition consisting of an extract ofRubia cordifolia can include inert or non-active compounds, such ascarriers or excipients.

The method can further include identifying a person in need of treatmentfor HIV/AIDS. Identifying a person in need of treatment can includetesting for the presence of HIV.

The extract or components of the extract of Rubia cordifolia can beformulated into pharmaceutical formulations suitable for parenteral ororal administration. As used herein, “pharmaceutical formulation” is acomposition of a pharmaceutically active drug, such as a biologicallyactive compound or extract, that is suitable for parenteral or oraladministration to a patient in need thereof and includes onlypharmaceutically acceptable excipients, diluents, carriers and adjuvantsthat are safe for parenteral administration to humans at theconcentrations used, under the same or similar standards as forexcipients, diluents, carriers and adjuvants deemed safe by the FederalDrug Administration or other foreign national authorities. An oralpharmaceutical formulation may be in the form of a capsule, tablet,solution, suspension, and/or syrups, and may also comprise a pluralityof granules, beads, powders, or pellets that may or may not beencapsulated. A parenteral pharmaceutical formulation may be in aready-to-use solution form, concentrated form, or a lyophilizedpreparation that may be reconstituted with a directed amount of diluentsuitable for parenteral injection such as water, salt solution, orbuffer solution. Examples of parenteral routes include subcutaneous,intramuscular, intravascular (including intraarterial or intravenous),intraperitoneal, intraorbital, retrobulbar, peribulbar, intranasal,intrapulmonary, intrathecal, intraventricular, intraspinal,intracisternal, intracapsular, intrasternal or intralesionaladministration.

Tablets prepared for oral administration will in one aspect containother materials such as binders, diluents, lubricants, disintegrants,fillers, stabilizers, surfactants, preservatives, coloring agents,flavoring agents and the like. Binders are used to impart cohesivequalities to a tablet, and thus ensure that the tablet remains intactafter compression. Suitable binder materials include, but are notlimited to, starch (including corn starch and pregelatinized starch),gelatin, sugars (including sucrose, glucose, dextrose and lactose),polyethylene glycol, propylene glycol, waxes, and natural and syntheticgums, e.g., acacia sodium alginate, polyvinylpyrrolidone, cellulosicpolymers (including hydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose, ethyl cellulose, hydroxyethylcellulose, and the like), and Veegum. Diluents are typically necessaryto increase bulk so that a practical size tablet is ultimately provided.Suitable diluents include dicalcium phosphate, calcium sulfate, lactose,cellulose, kaolin, mannitol, sodium chloride, dry starch and powderedsugar. Lubricants are used to facilitate tablet manufacture; examples ofsuitable lubricants include, for example, vegetable oils such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil, and oil oftheobroma, glycerin, magnesium stearate, calcium stearate, and stearicacid. Disintegrants are used to facilitate disintegration of the tablet,and are generally starches, clays, celluloses, algins, gums orcrosslinked polymers. Fillers include, for example, materials such assilicon dioxide, titanium dioxide, alumina, talc, kaolin, powderedcellulose and microcrystalline cellulose, as well as soluble materialssuch as mannitol, urea, sucrose, lactose, dextrose, sodium chloride andsorbitol. Stabilizers are used to inhibit or retard drug decompositionreactions that include, by way of example, oxidative reactions.Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents.

By a “therapeutically effective amount” of an extract of Rubiacordifolia is meant a sufficient amount of the extract or components ofthe extract to alleviate, modulate, or inhibit the negative or otherwiseill effects of HIV/AIDS infection. The extract of Rubia cordifolia canprovide one or more of the following benefits: boost general immunity;increase CD4 counts in HIV-infected patients; reduce HIV viral loads ininfected patients; and, reduce pathologies associated with HIV/AIDSprogression.

In some embodiments, the composition excludes extracts or powders of oneor more of the following plants: Scutellaria Baicalensis Georgi,Cordyceps sinensis, ginsenoside, Terminalia Belerica, AlstoniaScholaris, Piper longum, Corallocarpus Epigaeus, Tinospora Cordifolia,Aloevera India, Hemidesmus Indica, Phyllanthus Emblica, CinnamomumZeylamicum, Citrullus Colocynthis Scrad, Zingiber officinale, Foeniculumvalgare, Hyoscyamus niger, Succedania, Cyperus rotendus, Piper Longum,Commiphoria Mukul, Nordostachys, Withania Coagulans Dunal,Glycyrrhizaglabra, Pueraria Tuberosa, Santahim album, ArtemisiaSieversiana, Eleeoacrpus ganitreus, Phyllanthus niruri, BerberidaceaeBerberis Aristat D.C., Coriandrum Saticum, Vernonia anthelmintica, CarumCopticum, Azadirachta Indica A. Juss., Aristolochia Indica, Similaxglabra, Andropogum citrates Rose, Mentha arvensis, and Aegle Marmelos.

EXAMPLES Example 1 Preparation of Alcohol Extraction

10 milligrams of dry root powder of Rubia cordifolia was suspended in 1milliliter of 75% v/v ethanol. The suspension was rigorously vortexed.The suspension was allowed to stand at room temperature for at least 2hours and intermittently mixed with a vortex mixer. The supernatant wascollected to form the alcohol extract of Rubia cordifolia.

Example 2 Anti-HIV Activity Assay

The alcohol extract was tested in an in vitro HIV infection model. HIVinfectivity was measured by estimating the amount of HIV-1 p24 antigenproduction. Anti-HIV activity of the Rubia cordifolia extract was testedby measuring p24 antigen production and HIV-LTR expression.

Peripheral blood mononuclear cells (“PBMC”) from normal human subjectswere isolated using Ficoll gradient method. The cells were thenstimulated with phytoheam agglutinin for 48 hours. After 48 hours, thecells were harvested, washed, and resuspended in cell culture medium(RPMI 1640 medium available commercially from Sigma-Aldrich, St. Louis,Mo.) containing 10% fetal bovine serum and 2 μg/mL polybrene. The cellswere incubated for 30 minutes, and then infected with native HIV-1 IIIB(available from NIH AIDS Research and Reference Reagent Program,Germantown, Md.) at a concentration of 10³ tissue culture infectiousdose 50 (TCID₅₀) per mL for 2 hours.

The cells were then washed with phosphate buffered saline twice. 2×10⁶cells were plated in a 6-well plate. The alcohol extract of Rubiacordifolia was diluted to various concentrations and were added to theplated cells at 0.5 μg/mL, 1 μg/mL, 5 μg/mL, and 10 μg/mL of the alcoholextract of Rubia cordifolia. One well was left untreated and a controlof just ethanol was added to another well to serve as controls. Thispreparation method was used to test both for p24 antigen production andHIV-1 LTR gene expression.

p24 Antigen Production

For testing for p24 antigen production, the cells were incubated for 7days, and then harvested. The supernatant was collected for each well.The supernatant was tested for p24 antigen using a p24 ELISA kit(available commercially from ZeptoMatrix Corp., Buffalo, N.Y.). FIG. 1details the p24 antigen concentration for each well, including theresults from two controls (virus control and vehicle control). As theconcentration of the alcohol extract of Rubia cordifolia increased, theconcentration of p24 antigen decreased dramatically compared to thecontrols. Therefore, the extract of Rubia cordifolia significantlysuppressed p24 antigen production indicating inhibition of HIV-1.

HIV-1 LTR Gene Expression

For testing for HIV-1 LTR gene expression, the cells were incubated for24 hours, and then harvested. The RNA was extracted from the cellpellets. The RNA was reverse transcribed and followed by quantitative,real-time PCR against the LTR-RU5 and the housekeeping gene, β-actin, asinternal controls. FIG. 2 details the transcript accumulation index(“TAI”) for each well, including the results from two controls(untreated control and vehicle control). As the concentration of thealcohol extract of Rubia cordifolia increased, the TAI decreaseddramatically compared to the controls. Therefore, the extract of Rubiacordifolia significantly suppressed HIV-TLR gene expression in a dosedependent manner in HIV-infected PBMCs.

FIG. 3 is a comparison of 10 nM Azidothymidine with variousconcentrations of the extract of Rubia cordifolia. The extract atcertain concentrations demonstrated either equal or improved suppressionof the HIV-1 LTR gene expression.

Example 3a Cytotoxicity and Inhibition of HIV-1 p24 Production ofSamples 1-9

Various solvent extracts of the Rubia cordifolia plant, includingextracts of the roots, leaves, and stems, were prepared and tested foranti-HIV activity using an in vitro HIV-1 infection model. Solventsincluded isopropyl alcohol, water, and a combination of both. Samples 1,4, and 6 were aqueous extracts of the root of the Rubia cordifoliaplant. Sample 2 was an aqueous extract of the whole Rubia cordifoliaplant. Sample 3 was an aqueous extract of the stems of the Rubiacordifolia plant. Samples 5, 7, and 8 were hydro-alcoholic extracts ofthe root of the Rubia cordifolia plant. Sample 9 was an alcohol extractof the root of the Rubia cordifolia plant. PBMCs were infected withHIV-1 virus and subsequently cultured at two different concentrations ofRubia cordifolia extract, 10 μg/mL and 100 ng/mL. After 7 days ofincubation, non-specific cytotoxicity of these samples was assessed byXTT cytotoxicity assay (Table 1) with respect to virus control and cellculture supernatants were harvested to determine HIV-1 p24 antigenconcentration by ELISA (Table 2) with respect to virus control.

TABLE 1 Non-specific cytotoxicity of samples 1-9 % Cytotoxicity Sample10 μg/mL 100 ng/mL 1-6 No activity No activity 7 47 0 8 52.2 0 9 7.2 2.4

TABLE 2 Inhibition of HIV-1 p24 production of samples 1-9 % inhibitionof HIV-1 p24 antigen production Sample 10 μg/mL 100 ng/mL 1 0 0 2 45 0 331 19 4 56 0 5 78 17 6 80 0 7 78 25 8 89 0 9 74 25

Example 3b Cytotoxicity and Inhibition of HIV-1 p24 Production ofSamples 10-18

Various solvent extracts of the Rubia cordifolia plant, includingextracts of the roots, leaves, and stems, were prepared and tested foranti-HIV activity using an in vitro HIV-1 infection model. Solventsincluded isopropyl alcohol, water, and a combination of both. Sample 10was an aqueous extract of the whole Rubia cordifolia plant. Samples 11and 12 were aqueous extracts of the stems of the Rubia cordifolia plant.Sample 13-15 were aqueous extracts of the root of the Rubia cordifoliaplant. Sample 16-18 were alcoholic extracts of the root of the Rubiacordifolia plant. PBMCs were infected with HIV-1 virus and subsequentlycultured at two different concentrations of Rubia cordifolia extract, 10μg/mL and 100 ng/mL. After 7 days of incubation, non-specificcytotoxicity of these samples was assessed by XTT cytotoxicity assay(Table 3) with respect to virus control and cell culture supernatantswere harvested to determine HIV-1 p24 antigen concentration by ELISA(Table 4) with respect to virus control.

TABLE 3 Non-specific cytotoxicity of samples 10-18 % Cytotoxicity Sample10 μg/mL 100 ng/mL 10 0 0 11 26 10 12 36 19 13 45 25 14-15 No activityNo activity 16 17 0 17 97 15 18 99 31

TABLE 4 Inhibition of HIV-1 p24 production of samples 10-18 % inhibitionof HIV-1 p24 antigen production Sample 10 μg/mL 100 ng/mL 10 2.5 4.3 112.1 1.5 12 0.3 0 13 8.8 9.9 14 8.5 2.7 15 0 2.2 16 3.6 3 17 76.6 2.3 1884.1 22.9

The extract of Rubia cordifolia has demonstrated anti-HIV activity. Forexample, the extract of Rubia cordifolia can suppress HIV-1 LTR geneexpression. It can suppress p24 antigen production. The extract of Rubiacordifolia preferably provides greater than 45% inhibition of HIV-1 p24antigen production, more preferably, greater than 55% inhibition ofHIV-1 p24 antigen production, and most preferably, greater than 75%inhibition of HIV-1 p24 antigen production. The extract of Rubiacordifolia preferably provides greater than 45% non-specificcytotoxicity on peripheral blood mononuclear cells infected with HIV-1virus, more preferably, greater than 50% non-specific cytotoxicity, andmost preferably, greater than 75% non-specific cytotoxicity.

The foregoing description is given for clearness of understanding only,and no unnecessary limitations should be understood therefrom, asmodifications within the scope of the invention may be apparent to thosehaving ordinary skill in the art.

1. A method for treating a person with HIV/AIDS comprising:administering to the person a therapeutically effective amount of acomposition comprising an extract of Rubia cordifolia.
 2. The method ofclaim 1, wherein the composition consists of an extract of Rubiacordifolia.
 3. The method of claim 1, wherein the extract is an aqueousextract.
 4. The method of claim 1, wherein the extract is a water andalcohol extract.
 5. The method of claim 1, wherein the extract is analcohol extract.
 6. The method of claim 5, wherein the alcohol comprisesethanol.
 7. The method of claim 5, wherein the alcohol comprisesisopropyl alcohol.
 8. The method of claim 1, wherein the extract isprepared using water, organic solvent, or a mixture thereof.
 9. Themethod of claim 8, wherein the organic solvent is selected from thegroup consisting of low molecular weight alcohols, halogenatedhydrocarbons, organic ethers, low molecular weight esters, low molecularweight ketones, and mixtures thereof.
 10. The method of claim 1, whereinthe composition further comprises a pharmaceutically-acceptable carrieror excipient.
 11. The method of claim 1, wherein the compositionexcludes extracts from the group consisting of Scutellaria baicalensisGeorgi, Cordyceps sinensis, ginsenoside, Terminalia belerica, Alstoniascholaris, Piper iongum, Corallocarpus epigaeus, Tinospora cordifolia,Aloevera india, Hemidesmus indica, Phyllanthus emblica, Cinnamomumzeylamicum, Citrullus colocynthis Scrad, Zingiber officinale, Foeniculumvalgare, Hyoscyamus niger, Succedania, Cyperus rotendus, Piper longum,Commiphoria mukul, Nordostachys, Withania coagulans Dunal,Glycyrrhizaglabra, Pueraria tuberosa, Santahim album, Artemisiasieversiana, Eleeoacrpus ganitreus, Phyllanthus niruri, Berberidaceaeberberis Aristat D.C., Coriandrum saticum, Vernonia anthelmintica, Carumcopticum, Azadirachta indica A. fuss., Aristolochia indica, Similaxglabra, Andropogum citrates Rose, Mentha arvensis, Aegle marmelos.